Introduction

Attempts to make the polymerase chain reaction (PCR) quantitative have been made ever since the technique entered routine use (1). End point approaches are plagued by the problem of the plateau that occurs when reaction components become limiting and accumulating PCR products compete for polymerase binding (2). As a consequence of this plateau effect, a similar end point concentration may occur when samples contain quite different initial concentrations of nucleic acid (Fig. 1).

Fig. 1. End point methods of polymerase chain reaction are not suitable for the quantification of gene expression. A high-concentration sample (A) is indistinguishable from a lower-concentration sample (B) because of the plateau effect. In this example, sample B actually contains an initial concentration some 4000 times lower than sample A.

Fig. 1. End point methods of polymerase chain reaction are not suitable for the quantification of gene expression. A high-concentration sample (A) is indistinguishable from a lower-concentration sample (B) because of the plateau effect. In this example, sample B actually contains an initial concentration some 4000 times lower than sample A.

Early approaches to quantitative PCR (qPCR) were adopted infrequently, mainly owing to the numerous additional steps or reactions required, necessitating extensive optimization. Competitive PCR, for example, involves addition of known RNA standards to experimental samples before the reverse transcription (RT) step. These standards are typically the same sequence as that to be amplified, with an insertion or deletion enabling discrimination between standard and endogenous template, making this approach laborious, particularly when multiple transcripts are to be investigated (3).

The advent of real-time PCR has brought qPCR to the masses (4-7), and the number of publications featuring the technique has risen with an unerring similarity to the reaction itself. This technique has rapidly become the gold standard for the quantification of nucleic acids, offering exquisite sensitivity, a massive dynamic range (anything up to 8 log units), increased throughput of samples, and improved versatility when compared with traditional approaches such as Northern blotting or RNase protection assays. Consequently qPCR has become widely used for validating expression data produced from microarray experiments (8).

Real-time PCR (also referred to as kinetic PCR) combines improvements in fluorescent chemistry along with platforms typically consisting of a thermal cycler with laser (or other light source), optics and a detector system (typically a charge-coupled device camera, although a photomultiplier may be used), enabling single-sample imaging on a cycle-by-cycle basis (Fig. 2). In its simplest form, a real-time PCR platform may consist of a UV lamp as a light

Amplification Plot

Amplification Plot

Fig. 2. Real-time polymerase chain reaction setup. All real-time platforms essentially contain four components: a light source, a series of optics, a thermal cycler, and a recording device. The light source may be a white light source with filters or, more typically, a laser. The optics transmit the light from the source to the samples on the thermal cycler, then the fluorescence from the samples to detector. The recording device typically used is a charge-coupled device camera. The output is an amplification plot for each sample, plotting cycle number against fluorescence.

Fig. 2. Real-time polymerase chain reaction setup. All real-time platforms essentially contain four components: a light source, a series of optics, a thermal cycler, and a recording device. The light source may be a white light source with filters or, more typically, a laser. The optics transmit the light from the source to the samples on the thermal cycler, then the fluorescence from the samples to detector. The recording device typically used is a charge-coupled device camera. The output is an amplification plot for each sample, plotting cycle number against fluorescence.

source and charge-coupled device camera as a detector, and by inclusion of ethidium bromide in the actual reactions, the increasing ethidium bromide fluorescence may be monitored as the reaction progresses (9-10). More technologically advanced platforms offer better thermal profiles, improved optics, and more advanced chemistries, enabling improved precision and specificity (for details on real-time chemistries see refs. 4,7, and 11).

As the same principles underlie qPCR, this chapter will deal with general considerations rather than focusing on any one specific platform or chemistry (although, of course, drawing on personal experience).

Was this article helpful?

0 0

Post a comment